Cycloartane Triterpenoids from Euphorbia Macrostegia with their Cytotoxicity against MDA-MB48 and MCF-7 Cancer Cell Lines

The dried plant was extracted with dichloromethane and after defatting with hexane, transferred repeatedly on silica columns using dichloromethane-hexane and ethyl acetate-hexane as mobile phases. Finally the fractions were purified by high performance liquid chromatography using a Pack-Sil column and hexane: Ethyl acetate as mobile phase. The structures of the isolated compounds included: cycloart-25-ene-3β, 24-diol (1), cycloart-23(Z)-ene-3β, 25-diol (2), cycloart-23(E)-ene-3β, 25-diol (3), and 24-methylene-cycloart-3β-ol (4) were elucidated by 13C- and 1H-NMR as well as IR and by the aid of mass fragmentation pattern and comparing with the literature. The biological effects of the compounds were done by the MTT assay on two different cancer cell lines including MDA-MB48 and MCF-7. Among these compounds, cycloart-23(E)-ene-3β,25-diol (3) was the most active compound on MDA-MB468 cell line (LD50 = 2.05 μgmL− 1 ) and cycloart-23(Z)-ene-3β, 25-diol (2) was the most active compound on MCF-7 cell line (LD50 = 5.4 μgmL− 1).


Introduction
The incidence of cancer in human populations and the increasing need for anti-cancer drugs on the one hand and discovery of effective anticancer drugs, such as taxol, vincristin and vinblastin from plants. E.macrostegia as one of the endemic plants to Iran is the subject of this investigation. Euphorbia macrostegia (Persian wood spurge), belongs to the family Euphorbiaceae distributed mostly in central and west parts of Iran. Persian wood spurge is similar to the wood spurge (Euphorbia amygdaloides) and a rare species native of semimoist woods from south-eastern Europe through Asia Minor. In the Iranian traditional medicine, latex is used to treat warts. Despite their toxicity, the uses of Euphorbia species in traditional medicine in many parts of the world have a long history. They are used to treat inflammations and tumours (1). Previous investigation on the cytotoxicity assessment of E. macrostegia (2), has showed LD 50 values of 200, 425, and 390 water fall with elevation of 2130 m A.S.L. located in Yasooj, a city of Kohkilouyeh Va Boyer Ahmad province at Iran. It was identified by Department of Biology, Faculty of Science at University of Isfahan and a voucher specimen (#3340) was deposited in the herbarium of the Isfahan University (Iran).

Extraction and isolation
The air-dried plant material (2 Kg) was macerated with chloroform (20 L×3) at room temperature for 5 days. Filtration and in vacuo concentration resulted in a green gum (110 g), which was subjected on silica gel CC (hexane/ dichloromethane, 0→100) to several fractions: Fr 1-Fr 5. Inferred from TLC and 1 H-NMR, fraction Fr.1 and Fr.2 contained alkanes and fats, Fr.3 containing beta-sitosterol and fraction Fr. 4 and Fr.5 triterpenes. Fr.4 and Fr.5 were chromatographed on another normal column (hexane/acetone, 0→20). Finally triterpenes was further purified on HPLC using YMC-Pak-Sil column (250 × 20 mm) and hexane:ethylacetate (80:20) as mobile phase to μgmL − 1 for dichloromethane, ethyl acetate and acetone fractions, respectively while other fractions, remarked as noncytotoxic. Therefore, based on previous studies on cytotoxiciy effects of E. macrostegia and its fractions, the authors decided to investigate phytochemical contents of the dichloromethane extract of this plant as the most active fraction.

General experimental procedures
The NMR spectra were recorded on a Bruker Avance AV 400, using CDCl 3 as solvent. HPLC was carried out on a waters 515 using a YMC-Pack-Sil column (250 × 20 mm i.d.) and hexane: EtOAc as mobile phase. Chromatographic materials were silica gel (Merck Co., Germany). Thin layer chromatography detection was achieved by spraying the silica gel plates with cerium sulfate in 10% aq.H 2 SO 4 , followed by heating.

Plant material
Plant material was collected from Margoon

General experimental procedures
The NMR spectra were recorded on a Bruker Avance AV 400, using CDCl 3 as solvent.
HPLC was carried out on a waters 515 using a YMC-Pack-Sil column (250 × 20 mm i.d.) and hexane: EtOAc as mobile phase. Chromatographic materials were silica gel (Merck Co., Germany). Thin layer chromatography detection was achieved by spraying the silica gel plates with cerium sulfate in 10% aq.H 2 SO 4 , followed by heating.
Cell culture MCF-7 and MDA-MB468 human breast cancer cell lines were obtained from Pasteur Institute of Iran. The cell lines were grown adherently in RPMI-1640 media supplemented with10% fetal calf serum, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C in 5% CO 2 / 95% air. Cell  viability  was  determined  by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The MCF-7 and MDA-MB468 cells were seeded at 5 × 10 3 cells/well in 5% CO 2 at 37 °C in RPMI medium (containing 10% FBS, 100 units⁄mL penicillin and 100 μg⁄mL streptomycin) in 96well plates. After incubation overnight to allow for cell attachment, the RPMI medium in each well was replaced with by media containing various concentrations of compounds and incubated for 48 h. Afterwards, 20 μL of MTT (5 mg/mL in PBS) was added to each well and the cells were incubated for another 4 h at 37 °C. The supernatants were then aspirated carefully and 200 μL of dimethyl sulfoxide (DMSO) was added to each well. The plates were shaken for an additional 10 min and the absorbance values were read by the microplate reader (Bio-Rad, Hercules, CA, USA) at 570 nm. Cell viability was calculated as a percentage using the formula: (mean OD of treated cells /mean OD of control cells) ×100. The results expressed as percent of control cells which were not treated (3).

Statistical analysis
All samples were presented as mean ± SD for three measurements. Significance was attributed to p-values (P < 0.05) and the probability values obtained by the student t-test between sample and control data.

Result and Discussion
Compound 1, white crystals, showed the molecular formula of C 30 H 50 O 2 based on EI-MS m/z 442 and number and multiplicity of 13 C-NMR spectra. The six-degree of unsaturation and the 13 C-NMR data (Table 1) Regarding to these findings, and literature data (4), compound 1 identified as cycloart-25-en-3 β, 24-diol. It is also found in other Euphorbia species like E. aellenii (4), E. heteradena (5) and E. sessiliflora (6).
Compound 2, and 3 showed the molecular formula of C 30 H 50 O 2 based on positive EI-MS m/z 442 and in accordance with their number and the multiplicity of 13 C-NMR spectra (BB and DEPT). Their 1 H-NMR revealed six tertiary singlet methyls, one secondary methyl group, and a pair of doublets in the up-field area characteristic of cycloartane cyclopropane ring and one carbinolic proton related to 3(β)-OH group. In compound 2, in olefinic pair protons, δ In both compounds, downfield chemical shifts of two singlet methyl protons (Me-26, and Me-27) of the side chain atoms were in accordance with the second hydroxyl group on C-25 at δ C 70.8 and 68.2, respectively. Therefore, based on aforementioned data and complete agreements of 13 C-and 1 H-NMR with other reported data in literature (7; 8), compound 2 and 3 were identified as cycloart-23Z-ene-3β, 25-diol and cycloart-23E-ene-3β, 25-diol (Figure 1). They are also reported in Euphorbia spinidens (9), E. rigida (10), and E. humifusa (11). Compound 4, showed the molecular formula of C 31 H 52 O based on EI-MS m/z 440 and number and multiplicity of 13 C-NMR spectra. The six-degree of unsaturation and the 13 C-NMR data (Table 1) The potent cytotoxicity observed by compound 2 and 3 with double bound on C-23 suggested that the cytotoxicity activities of these compounds are related to the position of the olefinic or the hydroxyl group on side chain.